Didier Cussac

Cooperative Exosite-dependent Cleavage of Synaptobrevin by Tetanus Toxin Light Chain

The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH2 and COOH terminus of synaptobrevin have shown that the sequence 39-88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27-55 (S1) and S 82-93 (S2), to the synaptobrevin fragment S 56-81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S1 and S2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments and the determination of kinetic constants. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.

S32212, a Novel Serotonin Type 2C Receptor Inverse Agonist/α2-Adrenoceptor Antagonist and Potential Antidepressant: I. A Mechanistic Characterization

Although most antidepressants suppress serotonin (5-HT) and/or noradrenaline reuptake, blockade of 5-HT2C receptors and α2-adrenoceptors likewise enhances monoaminergic transmission. These sites are targeted by the urea derivative N- [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-1,2-dihydro-3-H-benzo[e]indole-3-carboxamide (S32212). S32212 was devoid of affinity for monoamine reuptake sites, yet displayed pronounced affinity (pKi, 8.2) for constitutively active human 5-HT2CINI (h5-HT2CINI) receptors, behaving as an inverse agonist in reducing basal Gαq activation, [3H]inositol-phosphate production, and the spontaneous association of h5-HT2CINI-Renilla luciferase receptors with β-arrestin2-yellow fluorescent protein. Furthermore, upon 18-h pretreatment, S32212 enhanced the plasma membrane expression of h5-HT2CINI receptors as visualized by confocal microscopy and quantified by enzyme-linked immunosorbent assay. Its actions were prevented by the neutral antagonist 6-chloro-5-methyl-N-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl]indoline-1-carboxamide (SB242,084), which also impeded the induction by long-term exposure to S32212 of otherwise absent Ca2+ mobilization in mouse cortical neurones. In vivo, S32212 blunted the inhibitory influence of the 5-HT2C agonist 2-(3-chlorobenzyloxy)-6-(1-piperazinyl)pyrazine (CP809,101) on ventrotegmental dopaminergic neurones. S32212 also blocked 5-HT-induced Gαq and phospholipase C activation at the h5-HT2A and, less potently, h5-HT2B receptors and suppressed the discriminative stimulus properties of the 5-HT2A agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane in rats. S32212 manifested marked affinity for human α2A- (pKi 7.2), α2B- (pKi 8.2), and α2C- (pKi 7.4) adrenoceptors, at which it abolished noradrenaline-induced recruitment of Gαi3, Gαo, adenylyl cyclase, and extracellular-regulated kinase1/2. Moreover, S32212 dose-dependently abolished the discriminative stimulus effects of the α2-adrenoceptor agonist (S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2′-(1′,2′,3′,4′-tetrahydronaphthalene)] (S18616). Finally, S32212 displayed negligible affinity for α1A-adrenoceptors, histamine H1 receptors, and muscarinic M1 receptors. In conclusion, S32212 behaves as an inverse agonist at h5-HT2C receptors and as an antagonist at human α2-adrenoceptors (and h5-HT2A receptors). Its promising profile in preclinical models potentially relevant to the treatment of depression is described in J Pharmacol Exp Ther 340:765–780, 2012.

Competitive interaction of 5-HT1A receptors with G-protein subtypes in CHO cells demonstrated by RNA interference

Following agonist action, G-protein-coupled receptors may exhibit differential coupling to G-proteins or second messenger pathways, supporting the notion of agonist-directed trafficking. To explore these mechanisms, we have designed and transfected synthetic siRNA duplexes to knockdown different G(α) subunits in Chinese hamster ovary (CHO) cells expressing human (h)5-hydroxytryptamine 1A receptors (CHO-h5-HT(1A)). siRNAs against G(αi2) and G(αi3) transfected alone or in combination caused a large decrease in the corresponding mRNA level (64-80%) and also at the protein level for G(αi3) (60-70%), whereas a non-specific siRNA showed no effect. In membranes of CHO-h5-HT(1A), 5-HT stimulated guanosine-5-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPγS) binding was differentially affected by transfection of siRNAs against G(αi) protein, siRNAs against G(αi2) inducing a more important decrease in the efficacy of 5-HT than transfection of siRNAs against G(αi3). The high potency component was abolished after transfection of siRNAs against G(αi3) and the lower potency component was suppressed after transfection of siRNAs against G(αi2). To directly investigate G(αi3) activation we used an antibody-capture/scintillation proximity assay. (+)8-OH-DPAT yielded bell-shaped curves for G(αi3) activation, a response that was abolished after transfection of siRNAs against G(αi3) protein. Interestingly, (+)8-OH-DPAT yielded a sigmoidal response when only G(αi3) protein was expressed. These data suggest that when efficacious agonists attain a high level of occupation of h5-HT(1A) receptors, a change occurs that induces coupling to G(αi2) protein and suppresses signalling through G(αi3) subunits.

Cellular BRET assay suggests a conformational rearrangement of preformed TrkB/Shc complexes following BDNF-dependent activation.

We developed a cellular Bioluminescent Resonance Energy Transfer (BRET) assay based on the interaction of TrkB fused to Renilla luciferase with the intracellular adaptor protein Shc fused to Enhanced Yellow Fluorescent Protein (EYFP). The TrkB agonist Brain Derived Neurotrophic Factor (BDNF) induced a maximum BRET signal as of 10 min with an EC(50) value of 1.4 nM, similar to the other endogenous agonists NT-3 and NT-4/5, 1.5 nM and 0.34 nM, respectively. Interestingly, measure of the BRET signal with increasing expression of Shc-EYFP, in the presence or absence of BDNF, suggested a conformational change of preformed TrkB/Shc complexes rather than Shc recruitment. Furthermore, the Y516F TrkB mutant deficient to bind Shc as well as the kinase-dead K572R TrkB mutant was unable to respond to BDNF and exhibited a lower basal BRET signal than that of the wild-type TrkB receptor, again suggesting a preformed complex with constitutive activity. The double YY706/707FF TrkB mutant in the kinase activation loop also showed reduced basal activity but surprisingly kept its capacity to enhance BDNF-induced interaction with Shc, though with less efficacy. The Trk selective kinase inhibitors K252a and BMS-9 blocked BDNF-induced BRET signal with similar potency (100-150 nM), the preferential c-Met inhibitor PF-2341006 being one order of magnitude less potent. Remarkably, in the absence of BDNF, K252a and BMS-9 also reduced basal activity to the level of the Y516F TrkB mutant, suggesting that these compounds were able to reduce the TrkB constitutive activity. BRET responses of mutants and to kinase inhibitors thus reveal a complex level of interaction between TrkB and Shc and suggest that this BRET assay could be of great utility to test blockers of TrkB signalling in a physiologically relevant context.

VIP-induced cross-talk between G-proteins in membranes from rat anterior pituitary cells

In order to study the activation mechanism of heterotrimeric G-proteins by agonist-liganded receptors, GTP gamma S binding to membranes was measured in rat adenohypophyseal cells after addition of dopamine (DA) or vasoactive intestinal peptide (VIP), which, respectively, inhibit and activate pituitary adenylyl cyclase. G-protein subunit present in anterior pituitary cells was characterized by either ADP-ribosylation catalysed by Bordetella pertussis and cholera toxins or by immunoblot using specific antisera. Binding of GTP gamma S was found to depend upon GTP gamma S and Mg2+ concentrations; it was sensitive to pretreatment of the cells with cholera and Bordetella pertussis toxins (IAP). DA increased binding of the nucleotide. Paradoxically, VIP decreased the rate of GTP gamma S binding; the effect was suppressed by prior treatment of the cells with either cholera toxin or IAP. VIP also increased [33P]ADPribose incorporation in Gi/Go-proteins catalysed by IAP. Forskolin was also able to decrease GTP gamma S binding, thus suggesting that the binding of forskolin with the adenylyl cyclase catalytic unit might activate Gs proteins through an increased interaction between Gs and adenylyl cyclase. Taken together, these results suggest that VIP, as well as forskolin, may both accelerate the activation of Gs and suppress the inhibitory effect of activated Gi/Go-proteins. Interactions between Gs and Gi/Go subunits mediated by beta gamma and/or adenylyl cyclase might thus result in a kinetic coupling of transduction pathways involving distinct G-proteins.

ADP-Ribosylation of Gαi and Gαo in Pituitary Cells Enhances their Recognition by Antibodies Directed Against their Carboxyl Termini

AbstractUsing antibodies raised against synthetic peptides of heterotrimeric GTP binding proteins, we demonstrate the presence of Gαs, Gαil,2, Gαi3, Gαo2, and Gβ subunits in pituitary cells. Pretreatment of pituitary cells with cholera toxin diminished the immunoreactivity of Gαs and this decrease was kinetically coupled to the rate of Gas ADP-ribosylation. ADP-ribosylation by islet activating protein (IAP or Bordetella pertussis toxin) of Gαi and Gαo enhanced their immunoreactivities to antibodies raised against synthetic decapeptides that correspond to the Gα carboxyl termini. Such enhancement was not observed when antibodies directed against the NH2-termini were used. These findings are consistent with the fact that ADP-ribosylation by IAP occurs on the cysteine located in the carboxyl terminal part of Gαi and Gαo. These observations mean that the kinetics and extent of Gi and Go ADP-ribosylation by IAP in whole pituitary cells and membrane preparations can be followed. It could be that ADP-ribosylatio...

Allosteric-type control of synaptobrevin cleavage by tetanus toxin light chain

The light chain of tetanus neurotoxin (TeNTL chain) has been shown to be endowed with zine endopeptidase activity, selectively directed towards the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein critically involved in neuroexocytosis. In previous reports, truncations at the NH2- and COOH-terminus of synaptobrevin have shown that the sequence 39–88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well-defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27–55 (S1) and S 82–93 (S2), to the synaptobrevin fragment S 56–81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S1 and S2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT probably involving a conformational change of the toxin. This could accound for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.